Acta Pharmacologica Sinica 2017: 1456–1465 © 2017 CPS and SIMM All rights reserved 1671-4083/17 www.nature.com/aps

Original Article

Novel mutations of TCIRG1 cause a malignant and mild phenotype of autosomal recessive (ARO) in four Chinese families

Xiao-ya ZHANG, Jin-wei HE, Wen-zhen FU, Chun WANG*, Zhen-lin ZHANG*

Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China

Abstract Human autosomal recessive osteopetrosis (ARO), also known as infantile malignant osteopetrosis, is a rare genetic bone disorder that often causes death. Mutations in T- immune regulator 1 (TCIRG1) are a frequent cause of human ARO. Six additional (TNFSF11, TNFRSF11A, CLCN7, OSTM1, SNX10, PLEKHM1) were also found to be associated with human ARO. In order to expand the mutation spectrum and clinical diversity for a better understanding of the ARO phenotype and to further investigate the clinical characteristics of benign subjects with ARO, we here report five individuals with ARO from four unrelated Chinese families. X-ray examination was conducted and bone turnover markers were assayed. The of T-cell immune regulator 1 (TCIRG1) was screened and analyzed. -induced osteoclasts were prepared and their resorption ability was studied in vitro. We identified five novel mutations (c.66delC, c.1020+1_1020+5dup, c.2181C>A, c.2236+6T>G, c.692delA) in these patients. Four patients displayed a malignant phenotype, three of them died, and one who received transplantation survived. The remaining one, a 24-year- old male from a consanguineous family, was diagnosed based on radiological findings but presented no neurological or hematological defects. He was homozygous for c.2236+6T>G in 18; this mutation influenced the splicing process. Anin vitro functional study of this novel splicing defect showed no resorption pits on dentine slices. TCIRG1-dependent osteopetrosis with a mild clinical course was observed for the first time in Chinese population. The present findings add to the wide range of phenotypes of Chinese patients with TCIRG1-dependent ARO and enrich the database of TCIRG1 mutations.

Keywords: autosomal recessive osteopetrosis; TCIRG1; malignant; mild phenotype; Chinese family

Acta Pharmacologica Sinica (2017) 38: 1456–1465; doi: 10.1038/aps.2017.108; published online 17 Aug 2017

Introduction cavity and nerve compression. Li et al showed that the tar- Human autosomal recessive osteopetrosis (ARO) represents a geted disruption of Atp6i in mice resulted in severe osteo- group of inherited bone disorders characterized by diffusely petrosis[4]. Then, in 2000, Frattini et al elucidated for the first increased bone density due to the failure of bone resorption time that mutations in T-cell immune regulator 1 (TCIRG1) by osteoclasts[1]. ARO is a rare disease that has an incidence are a frequent cause of human ARO[5]. Subsequently, molecu- of 1 in 250 000 births[2], but the incidence is particularly high lar analysis has revealed that six additional genes (TNFSF11, in specific geographic regions (eg, Costa Rica, the Middle East, TNFRSF11A, CLCN7, OSTM1, SNX10, PLEKHM1) are associ- the Chuvash Republic of Russia and the Province of Västerbot- ated with human ARO. Mutations in TCIRG1 are responsible ten in northern Sweden). This distribution is attributed to the for more than 50% of ARO-affected individuals[6], demon- founder effect, geographic isolation or high parental consan- strating the crucial role of the V-ATPase in osteoclast func- guinity[3]. ARO renders bones more susceptible to hematologi- tion. Despite rapid development in the understanding of the cal impairment and secondary neurological deficit (blindness pathogenesis of osteopetrotic conditions, the genetic basis of or deafness), which are caused by a decreased bone marrow approximately 30% of cases remains to be elucidated[2]. Sobacchi et al and others have provided insight on a wide variety of TCIRG1 mutations, including missense, non- *To whom correspondence should be addressed. E-mail [email protected] (Zhen-lin ZHANG); sense, small deletions/insertions, splice-site mutations, large [5, 7-10] [email protected] (Chun WANG) genomic deletions and intronic mutations . However, Received 2017-01-19 Accepted 2017-05-11 data concerning Chinese TCIRG1-deficient ARO patients are www.chinaphar.com Zhang XY et al 1457 relatively limited, and include mainly reports of individual Materials and methods mutations[11, 12]. Here, we described a relatively large sample Subjects and samples of affected Chinese individuals with variable clinical severi- This study was approved by the Ethics Committee of the ties, and we focused on the identification of the molecular defect Shanghai Jiao Tong University Affiliated Sixth People’s Hospi- and osteoclast functional characterization of a 24-year-old male tal. The pedigrees of these families are shown in Figure 1, and who came from a consanguineous family. Moreover, the mild the major clinical findings are summarized in Table 1. phenotype of osteopetrosis was observed for the first time in Chinese population. To date, the only cure is hematopoietic stem Family 1 cell transplantation (HSCT), and we sincerely hope that more A 10-month-old proband (Pt 1, II1) (Figure 1A) was referred to patients are reported and further studies are performed. us for genetic diagnosis with clinical manifestations of ARO.

Figure 1. (A) Pedigrees of the four Chinese families with TCIRG1-dependent ARO. Arrows indicate the probands. Filled black symbols refer to patients with ARO. Half-black symbols represent healthy carriers with a heterozygous mutation. (B) Location and sequencing of pathogenic TCIRG1 mutations. Family 1: c.1020+1_1020+5dup and c.66delc; Family 2: c.1213G>A and c.2181C>A; Family 3: c.292delA and c.909C>A; Family 4: c.2236+6T>G; (C). p.Tyr23ThrfsX4 (Y23), p.Lys231ArgfsX48 (K231), p.Gly405Arg (G405), p.Cys727X (C727) occur at a highly conserved position in TCIRG1, as shown by a comparison the corresponding sequence of 7 vertebrates.

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Table 1. Laboratory data of TCIRG1-dependent patients.

Pt 1 Pt 2A Pt 3 Pt 4 Ref range

Age 9 months 53 d 7 months 24 years Calcium (mmol/L) - 2.05 1.36 2.60 2.08–2.60# Phosphorus (mmol/L) - 0.82 1.96 1.23 0.80–1.60# PTH (pg/mL) - - - 53.38 15.00–65.00 25(OH)D (ng/mL) - - - 22.98 11.11–34.43* ALP (U/L) - 767 - 71 15–112# LDH (U/L) - 829 1927 213 150–360 β-CTX (ng/L) - - - 707 100–612* OC (ng/mL) - - - 31.25 5.58–28.62* WBC (×109/L) 24 23.65 13.1 4.0 3.7–10 RBC (×1012/L) 2.8 3.23 3.45 5.19 3.68–5.74 PLT (×109/L) 47 35 126 112 90–320 HGB (g/L) 71 104 91 150 113–172

PTH, parathyroid hormone. * reference values were calculated from our previous study. # reference range for adults; pediatric reference values of calcium, phosphorus, ALP for 1–3 years old were 2.13–2.70, 1.4–2.3, 58–400 respectively. - data not available.

He was the only child of unrelated parents and was born at chondral junctions at the ends of the ribs and a widened growth full-term via caesarean section. His tooth did not erupt when plate. Further inspection confirmed osteopetrosis (Figure 2A). he was referred to us, and he was smaller than his contempo- Blood tests indicated decreased serum calcium and phospho- raries. His height was 62 cm (normal range: 71.0–76.3 cm) and rus. Ultrasonography revealed that his liver was 24 cm below weight was 7.0 kg (normal range: 8.6–10.6 kg). At approxi- his right costae. Pathological features, such as overall growth mately 9 months old, he suffered from acute infection with retardation, delayed tooth eruption, tooth loss, poor vision and high fever, and routine blood tests showed clearly elevated recurrent infections, became evident, and finally he died of leucocytes and decreased red cells, platelets and hemoglobin. multiple organ failure at 4 years old. His height was 92.0 cm Abdominal ultrasonography detected an enlarged spleen (normal range: 98.7–107.2 cm) and weight was 12.2 kg (normal and liver. His liver margin was 4.0 cm below the right costae range: 14.8–18.7 kg). The patient had only four teeth at the time and the spleen margin was 6 cm below the left costae. Chest of death. Pt 2B, who was two years younger than Pt 2A, experi- radiography confirmed generalized osteopetrosis, with a wid- enced a normal childbirth. At approximately one month old, his ened growth plate widened and nodular rib ends. Magnetic anxious parents took him to the hospital for physical examina- resonance imaging (MRI) showed hydrocephalus as well as tion. Radiographs revealed elevated bone mineral density. He a downward displacement of the cerebellum. After carefully suffered from growth retardation [height 75 cm (normal range: evaluating the potential risks, surgeons successfully treated 84.3–91.0 cm) and decreased weight 10.5 kg (normal range: 11.2– the patient with a ventriculo-peritoneal shunt for hydrocepha- 14.0 kg) at two years old], anemia, and recurrent infections. His lus. The patient received bone marrow transplantation (BMT) first tooth erupted at approximately one year old. He could from two HLA-antigen mismatched related donors at approxi- not walk. He died of bone marrow failure at 2 years old. mately 13 months old. A combination of fludarabine, busulfan and cyclophosphamide was used as the conditioning regimen; Family 3 he received approximately 29.9×108/kg mononuclear cells and Patient 3 (Pt 3, II1, Figure 1A), a male, died of bone marrow 10.9×106/kg CD34+ cells. His illness was complicated by con- failure at approximately 7 months old. His parents came to comitant acute graft-versus-host-disease (GVHD), which was us for genetic counseling with a small piece of dried umbili- accompanied by diarrhea and skin rash. He is alive as of the cal cord 15 months after his death. The proband was born to time of this report. a non-consanguineous couple after an uneventful delivery, but he was admitted to a local hospital due to convulsion at Family 2 6 months. Blood tests revealed decreased serum calcium and Patients 2A and 2B (Pt 2A, II1; Pt 2B, II2) (Figure 1A) were two hemoglobin. Total body radiographs (Figure 2B) confirmed deceased, affected siblings born from a non-consanguineous bone sclerosis with a widened growth plate. During hospital- family. Pt 2A, a male, was born via natural childbirth and ization, he developed bronchopneumonia, which worsened was suffering from fever, cough, nasal congestion and runny his outcome. No further inspections, such as abdominal ultra- nose. He was admitted to a local hospital at approximately 20 sonography or optic nerve computerized tomographic (CT) d old; chest X-ray revealed typical rickets-like nodular costo- scanning, were performed before he died.

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Figure 2. Representative X-rays of Pt 2A, Pt 3, Pt 4, showing the diffusely increased bone density. (A) Pt 2A at 2 years, notice the increased vertebra and rib density as well as typical rickets-like nodular costochondral junctions at the end of ribs; (B) Pt 3 at 6 months, radiograph of the legs show bone sclerosis along with fraying and splaying of metaphyses, growth plate widening and diffuse periosteal reaction (arrows indicated); C, D, E, F, G are pictures of Pt 4. (C) The pelvic bones, as well as femoral head, proximal femora demonstrate high bone density; (D) Lateral cephalogram indicates sclerotic skull base and cervical vertebra; (E) Unlike the typical “rugger-jersey appearance” of CLCN7-dependent ADO type, this standard radiograph of the spine (anteroposterior) showing the homogeneous sclerosis of the vertebra; (F) Broken bone and external fixation when Pt 4 was 16 years old; (G) The obliteration of marrow cavity and bone deformity of right distal femur resulting from non-perfect healing and fracture remodeling.

Family 4 chloroform and isopropyl alcohol precipitation. The DNA A 24-year-old proband (Pt 4, IV2) (Figure 1A) was the second of Pt 3 was extracted from his dried umbilical cord using the child of unaffected first-cousin parents. His older sister was QIAamp DNA Investigator kit (Qiagen, Hilden, Germany). healthy. His past medical history included tooth decay (eigh- Molecular analysis was performed by amplification and teen years old, three molars of the right maxillary) and dental direct sequencing of and intron- boundaries as repair. He was initially evaluated at 16 years old because of a previously described[14]. The sequences of the TCIRG1 prim- right distal femur fracture from jumping on the ground. The ers used are available in Supplementary Table 1. After the X-ray demonstrated a broken bone with an almost vanished peripheral blood mononuclear cells of Pt 4 were lysed in medullary cavity because of sclerotic bone change (Figure TRIzol, mRNA was isolated and reverse transcribed. The 2C-2G). After the fracture healed, he did not receive any effect of intronic variants on TCIRG1 transcript processing specific therapy, attended school as usual, avoided physi- was investigated at the cDNA level with the forward primer cal activity, and did not experience fracture, hematological 5´-CCTACACGACGCTCTTCCGATCTAAAACAAGGC- or neurological defects. At the age of 24, he arrived at our CGGGTTGCTG-3´ located in exon 17 and the reverse primer clinic with back pain; his height was 159 cm and weight was 5´-TCAGACGTGTGCTCTTCCGATCTAAGGCTGAGA- 50.8 kg. Peripheral blood cells, calcium, phosphorus, alkaline GTCCCTCCATCAC-3´ in exon 19 (NM_006019.3) using the phosphatase, parathyroid hormone, and 25-hydroxy vitamin following thermocycling conditions: initial denaturation step D levels were normal. However, bone turnover markers, such at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 as osteocalcin and beta C-terminal cross-linked telopeptides °C for 15 s, annealing at 60 °C for 1 min and finally a dissocia- of type I collagen (β-CTX), were slightly elevated based on tion stage. Paired-end sequencing was performed on the Illu- the reference ranges previously established[13] (Table 1). The mina miseq2*250 platform with a sequencing depth of 5000×. Z scores of lumbar 1-4, femoral neck and total hip were +16.6, The minimum data collected were 0.2 M. +18.4, and +16.6, respectively. A thorough examination of the 3-generation pedigree excluded any disease symptoms in rela- Whole exome sequencing of Pt 4 tives. Currently, the patient is still in follow-up. We sequenced the exome of the single affected individual (Figure 1A, family 4, IV2) from a consanguineous family. Molecular studies Exon-enriched DNA from the proband was sequenced by the DNA was extracted from peripheral white blood cells by Illumina Genome Analyzer II platform according to the manu- proteinase K digestion followed by purification with phenol/ facturer’s instructions. Raw image files were processed with

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Illumina Base Calling Software 1.7 with default parameters, observation, the discs were cleaned ultrasonically with dis- and the sequences of each individual were reported as 90 bp tilled water (5 min×3 times) for scanning electron microscopy paired-end reads. Sequence reads were mapped to a reference (SEM). The specimens were fixed in 2.5% glutaraldehyde in genome (UCSC Genome Brower hg18 assembly) with SOAP2 0.1 mol/L phosphate buffer for 2 h at 4 °C, washed three times (BGI-Shenzhen)[15-17]. The SOAP SNP results were filtered in the same buffer and post-fixed in 1% osmium tetroxide for 2 according to a standard workflow for exome sequencing. h at 4 °C. The samples were then washed 3 times in 0.1 mol/L buffer, dehydrated through graded ethanol washes (50%, 70%, Analysis of in vitro osteoclast function of Pt 4 90%, 100%×3, each for 15 min), then transferred to mixtures of Peripheral blood mononuclear cells (PBMNCs) were isolated 100% ethanol and amyl acetate at different volume ratios (2:1, from 10 mL of blood from Pt 4 and an age-matched healthy 1:2), and finished in pure amyl acetate. Slices were prepared ® male according to Lympholyte -H (Cedarlane Laboratories by the CO2 critical point drying method and coated with gold Ltd) instructions. Then, PBMNCs were diluted with 1-2 mL before being examined in a Hitachi SU8010 SEM at 10 kV. α-Minimum Essential Medium (MEM; 10% fetal calf serum, 2 mmol/L L-glutamine, 100 U/mL penicillin, 0.1 mg/mL strep- Results tomycin) containing 20 ng/mL recombinant human macro- Genetic findings phage colony-stimulating factor (rhM-CSF; R&D Systems) and The results of the mutations are summarized in Figure 1B. seeded in 96-well plates and 6-well plates (2×105 and 2.5×106 Mutations of Y23, K231, G405, and C727 occur at a highly con- cells/well, respectively) for 9 d, with a complete change of cul- served position of TCIRG1 among vertebrates (Figure 1C) and ture medium every 3 d. Then, M-CSF-dependent macrophages were predicted to have pathogenic effects by ACMG/AMP in 6-well plates were rinsed with cold PBS and scraped into standards and guidelines[19] (Table 2). fresh medium after incubation with 0.1% (1 mg/mL) trypsin In family 1, we detected compound heterozygous muta- for 15 min. After the cells were centrifuged (300×g, 5 min) and tions: a heterozygous c.1020+1_1020+5dup in intron 9 that resuspended in fresh medium, they were seeded onto 96-well was inherited from the father and another mutation in exon plates and 9-mm discs of dentine in 24-well plates (2×104 and 2, c.66delC, which resulted in a sequence change after amino 1×105 cells/well). After plating, all cells were incubated with acid 23 and introduced a new stop codon after 4 new amino 20 ng/mL rhM-CSF and 100 ng/mL receptor activator of acids (p.Tyr23ThrfsX4). The latter was a de novo mutation. nuclear factor-κB ligand (RANKL, PeproTech) for 11 d, with In family 2, the proband carried compound heterozygous medium replacement every 3 d. Cells were subsequently fixed mutations: a heterozygous c.1213G>A mutation in exon 11 in paraformaldehyde for light microscopic analysis or glutar- inherited from his mother that resulted in a glycine-to-arginine aldehyde for electron microscopic analysis. Osteoclasts were substitution at position 405 (p.Gly405Arg) and another het- also stained for tartrate-resistant acid phosphatase (TRAP) erozygous mutation in exon 18, c.2181C>A, inherited from using naphthol-ASBI-phosphate substrate[18] (Sigma-Aldrich, his father, which generated a stop codon at position 727 Leukocyte Acid Phosphatase, 387A-1KT) and analyzed using (p.Cys727X). In combination with the late younger brother’s an inverted microscope equipped with a CCD camera (DMI clinical features and family history, we reasonably suspected 3000B/DFC 450C, Leica). Resorption pits on fixed dentine that the younger brother shared the same heterozygous muta- discs were subjected to ultrasonic cleaning with distilled water tions with the proband. at 50 kHz for 5 min, dehydrated with an ethanol gradient In family 3, DNA extracted from umbilical cord indicated (40%, 70%, 90%, 95%, 100%) for 30 s and left to dry naturally. that the deceased proband was a compound heterozygote: a They were then stained with 1% toluidine blue at room tem- heterozygous c.962delA in exon 7 inherited from his father led perature for 5 min and washed with distilled water. After to a sequence change after amino acid 231, generating a new

Table 2. Molecular findings identified ofTCIRG1 -dependent patients.

Patient Location cDNA changea Effect Variant classificationb

Pt 1 Exon 2, DEL c.66delC p.Tyr23ThrfsX4 Pathogenic (Ib) Intron 9, DUP c.1020+1_1020+5dup Putative aberrant splicing Pathogenic (Ib) Pt 2A Exon 11, missense c.1213G>A p.Gly405Arg Pathogenic (Ia) Exon 18, nonsense c.2181C>A p.Cys727X Pathogenic (Ia) Pt 3 Exon 7, DEL c.692delA p.Lys231ArgfsX48 Pathogenic (Ia) Exon 9, nonsense c.909C>A p.Tyr303X Pathogenic (Ia) Pt 4 Intron 18 c.2236+6T>G Putative aberrant splicing Pathogenic (Ia)

aAccession number of the TCIRG1 variant cDNA:NM_006019 the nomenclature used nucleotide+1 as A of the ATG-translation initiation codon, intron change used the number of the last nucleotide of the proceeding exon plus the position in the intron. b variant classification was generated according to the new ACMG/AMP standards and guidelines (Genet Med 2015; 17: 405-24).

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stop codon after 48 new amino acids (p.Lys231ArgfsX48); the firm three major forms of transcripts. Approximately 60938 other heterozygous mutation, c.909 C>A in exon 9, which was transcripts were missing exon 18 and accounted for 82.1% inherited from his mother, resulted a stop codon at position of transcripts, 1358 transcripts displayed a normal sequence 303 (p.Tyr303X). and accounted for 1.8%, and approximately 10332 transcripts In family 4, molecular analysis initially focused on common retained intron 18 and accounted for 13.9% (Figure 3B, 3C). genes; however, no obvious defects could be identified. We proceeded with whole-exome sequencing of Pt 4 using previ- Osteoclast differentiation and function of Pt 4 ously adopted methods[20]. Because the mode of inheritance We analyzed the ability of osteoclasts to differentiate in vitro is likely recessive in this consanguineous family, we concen- from isolated from the nontransplanted TCIRG1- trated on homozygous nonsynonymous variants in coding mutated patient with relatively mild clinical symptoms, com- sequences. We preliminarily screened osteopetrosis-corre- pared to an age-matched healthy male. The growth and num- lated genes (LRP5, TNFSF11, TNFRSF11A, CLCN7, OSTM1, ber of osteoclasts were assessed by TRAP staining. TRAP+ SNX10, PLEKHM1, CA2), but there were no mutations in these multinucleated cell numbers and cell morphology between the genes. Therefore, we returned to the classical Wnt/β-catenin healthy control and the patient are similar (Figure 4). More- OPG/RANKL/RANK pathway and genes closely correlated over, when cultured on dentine, osteoclasts from the healthy with osteoclast function. We sequenced a dozen genes but control resorbed large pits that were observed using light had no success in finding the correct mutation. Finally, we microscopy (toluidine blue staining) and SEM, but no absorp- re-visited CLCN7 and TCIRG1 and examined the 3’ UTRs of tion lacunae were detected in the patient cultures (Figure 4). CLCN7 and TCIRG1, which may influence . Noticeably, a single nucleotide change in intron 18 of TCIRG1 Discussion (NM_006019.3, c.2236+6T>G) was annotated in the homozy- Human ARO is a rare genetic bone disease that is character- gous state (G/G), and other family members all presented ized by generalized increased bone density and a variety of heterozygous (T/G) for this . Then, RNA extracted from heterogeneous symptoms, including hematological and neural his whole blood was reverse transcribed into cDNA. Gel elec- defects with diverse severity[21]. TCIRG1 is located on chromo- trophoresis of the cDNA PCR products spanning exon 17 to 19 some 11q13, encodes the osteoclast-specific 116 kD subunit of clearly presented two abnormal transcripts (Figure 3A). Next- the vacuolar , contains 20 exons, and mediates generation paired-end sequencing generated a total of 74180 H+ transport into the resorption lacunae, where a low pH is a read pairs. By identifying the read pairs, we were able to con- prerequisite for the dissolution of hydroxyapatite crystals[22].

Figure 3. Gel electrophoresis of the cDNA PCR products showing the presence of two abnormal bands in Pt 4, the band 675 bp represented the transcripts which retained intron 18, 255 bp represented the transcripts which skipped exon 18, normal band was not seen; (B) Schematic representation of the relative region of the TCIRG1 gene, highlighting the position of the mutation identified in intron 18, the nucleotide change (c.2236+6T>G) found in Pt 4 is directed to lower line written in capital letter. (B) Structure of the major three types of aberrant transcripts found in Pt 4, the first one was spliced with missed exon 18, the second one was the normal transcript, while the third one retained intron 18.

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Figure 4. Osteoclast generation and bone resorptive activity in vitro from individual with TCIRG1-defected ARO. PBMNCs from peripheral blood mononuclear cells of Pt 4 and one age-matched male control were concurrent cultured for 9 d with rhM-CSF, then digested and seeded on dentine discs in the presence of M-CSF and RANKL for 11 d. (A) Cells (magnification: 200×) in 96-well plates stained for TRAP, showing abundant multinucleated, TRAP-positive cells in both control and Pt 4; (B, C) Lacunae on dentine slices observed by (B) light microscopy with toluidine blue staining, 100× and (C) scanning electron microscopy. Light micrograph of the control showing varied shape of resorption lacunae scatter on the dentine, SEM image indicates circular, elliptical, sausage and irregular shaped resorption pits with typical punched-out osteoclastic excavations and exposure of collagen fibres. Bone cultures of osteopetrotic PBMNCs show no sign of absorption. Scale bar, 100 μm in the bottom of the photographs. (D) TRAP-positive multinucleated osteoclast cell numbers are shown (we calculated 5 culture holes in 96-well plates, repeated 3 times), statistical difference between two group of 96- well plates were determined by the Student’s t test and analyzed by a one-way ANOVA followed by Student Neuman Keuls (S-N-K) testing.

Generally, patients with defective TCIRG1 display life-threat- notypes and typical hematological abnormalities in infancy, ening symptoms in their early lives and urgently need HSCT. Pt 4, from a consanguineous family, did not display serious In this study, we described five patients (from four unre- symptoms until high school. His disease progression was lated families) who suffered from osteopetrosis. We discov- relatively slow and benign, suggesting the existence of inter- ered seven different TCIRG1 mutations (Figure 1B), of which mediate cases. In family 1, family 2 and family 3, compound five mutations (c.66delC, c.1020+1_1020+5dup, c.2181C>A, heterozygous mutations of TCIRG1 contributed to the early c.2236+6T>G, c.692delA) were novel based on the Exome onset of osteopetrosis. The de novo mutation carried by Pt 1 Aggregation Consortium (ExAC) database and the Human was suspected to be the result of an alteration in a germ cell or Gene Mutation Database (HGMD). Whereas Pt 1, Pt 2A, Pt in the fertilized egg itself. Remarkably, the affected children 2B and Pt 3 all manifested with severe osteopetrotic bone phe- from family 2 and family 3 shared two point mutations, one

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inherited from the mother and the other from the father. The and phosphorus metabolism, with related osteopetrorickets, typical rickets-like nodular costochondral junctions at the ends but the mild phenotype did not present these characteristics. of ribs and the widened growth plate, also termed osteopetror- The BTMs of the malignant patients were not available, but the ickets, presented in the malignant affected pediatric patients, BTMs of the mild phenotype were normal or slightly elevated. but not the mildly affected patient. Sobacchi et al did not The patients of the mild form with known mutations all had detect osteopetrorickets in affected individuals with the mild splicing defects that exerted an influence on the latter part of form either. Osteopetrorickets was initially suspected to be a the TCIRG1 . This finding may indicate that the last paradoxical complication of osteopetrosis resulting from the 5 exons of the TCIRG1 protein are not quite as important as inability of osteoclasts to maintain a normal calcium-phospho- the N-terminal portion, or that they are partially compensated rus balance in the extracellular fluid[23]. In this study, patients by the limited amount of normal transcripts. Interestingly, with osteopetrorickets displayed abnormal metabolism of cal- patients with this mild form with extremely dense but brittle cium and phosphorus. bones and an almost vanished medullary cavity did not result The identification of the mutation in Pt 4 was a tortuous in normocytic anemia or thrombocytopenia, which highlights course. Further verification in family members and transcript the strong compensatory ability of the hematopoietic tissue. analysis confirmed our suspicion that two different aberrant There are also common features between the severe and mild transcripts were produced, the most abundant being the tran- phenotypes, such as dental abnormalities and dense bone. scripts missing exon 18, while the second were transcripts However, many complex issues remain to be resolved; for that retained intron 18. We also noticed that a small number example, how do the transcripts function, in what quantity, do of normal transcripts were generated, which allowed a small they function alone or are they modified by other genes? How amount of protein with partial activity to compensate will this benign form progress in the following years, slowly, for part of the osteoclast functional defect in vivo. This find- as before, or will complications, such as nerve compression, ing would explain the overall benign form of ARO with slow occur? progression. Nevertheless, in vitro osteoclast function tests The incidence of osteopetrosis related to the Chinese popu- showed normal quantities and morphology of cells, but they lation has not yet been reported due to the immense popula- were still unable to resorb bone when exposed to rhM-CSF tion base. However, the number of individuals who suffer and RANKL co-cultures, confirming that the vacuolar proton from the disease cannot be ignored. HSCT, the most widely pump plays a fundamental role in the late stage of osteoclast adopted treatment abroad, has been shown to be the only physiology. These intrinsic defects were unable to be rescued cure for the prevention and reversal of bone manifestations[3]. by rhM-CSF and RANKL. However, because HSCT does not improve already existing Recently, Sobacchi et al reported that an incomplete splic- neurological sequelae[26], it should be performed as soon as ing defect in TCIRG1 led to benign, recessive osteopetrosis possible. Unfortunately, very few domestic hospitals that in an 8-year-old girl. This result revealed that a mild form are capable of performing HSCT have launched therapeutic of TCIRG1-dependent ARO does exist[9]. Then, the team fur- programs for patients with osteopetrosis. In 2011 and 2012, at ther discovered 3 different single nucleotide changes deeply meetings of the IEWP-EBMT (Inborn Error Working Party of embedded in intron 15 that were predicted to impact the splic- the European Group for Blood and Marrow Transplantation), ing process of the neighboring exons, which suggested that a survey showed that 10 patients younger than 10 months of this region may be a hot spot region for mutation[10]. Notably, age survived HSCT, but almost all patients experienced com- in the present study, a mutation in the splice site c.2236+6T>G plications of graft rejection or autologous engraftment recon- has not been described previously, but a homozygous stitution, even when using cells from an HLA-haploidentical c.2236+1G>A near this location has been reported[24]. The donor[27]. Ordinary families in China can hardly afford the affected male was born into a consanguineous Muslim fam- cost of treatment; thus, many patients have not been properly ily and presented with noisy breathing, prominent eyes, and treated or received correct genetic counseling. The combina- bleeding spots early in life. He was diagnosed at 18 months, tion of molecular biology and amniocentesis promotes the and he had two older affected siblings who died in infancy. application of prenatal diagnosis, which facilitates early inter- Other adjacent homozygous mutations that may exert an influ- vention for the already suffering family based on the known ence on the splicing process have also been investigated[6, 25], pathogenic mutations. but these changes led to infantile malignant osteopetrosis. There were several limitations in our study. First, a single Variants affecting splice sites represent a high percentage case of the benign form of TCIRG1-induced ARO is not suffi- of the molecular abnormalities in the TCIRG1 gene (44.2%), cient to summarize all properties of this mild phenotype. Sec- among which the invariant GT/AG dinucleotide is 30.4%[25]. ond, some serum parameters, such as BTMs, were not avail- The mutations discussed here are located close to the canoni- able. In addition, we did not perform studies on the work- cal splice sites, but their functional impact generated different ing mechanism of this mild phenotype and more in-depth outcomes. research is needed. The comparisons between the malignant form in this study In conclusion, we described a relatively large sample of and the known mild form are listed in Table 3. It is notewor- TCIRG1-dependent ARO, reported for the first time a mild thy that malignant patients displayed a disorder of calcium phenotype in the Chinese population, and enriched the data-

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Table 3. Comparisons of TCIRG1-dependent malignant phenotype in this study with known mild phenotype.

Malignant phenotype Mild phenotype

Pt 1 Pt 2A Pt 3 Pt 4 5 6 7 8 Geographical origin China China China China NA Italian Italian Italian Rererence this study this study this study this study 9 10 10 10 Consanguinity No No No Yes Yes Yes Yes No Mutational Exon 2, Exon 11, Exon 7, Exon 18, Intron 15, Intron 15, Intron 15, Intron 15, location Intron 9 Exon 18 Exon 9 SD SD SD SD SD; Exon 14 Status ComHet ComHet ComHet Homo Homo Homo Homo ComHet Age of onset 9 ms 53 d 7 ms 24 years 3 years 6 years 2 years 4 years Outcome Alive Died Died Alive Alive Alive Alive Alive Osteopetrosis Yes Yes Yes Yes Yes Yes Yes Yes Ca, P disorder NA Yes Yes No No No No No Osteopetrorickets Yes Yes Yes No No No No No Hepatosplenomegaly Yes Yes NA No No Yes No No Anemia Yes Yes Yes No No Mild No No Neurological defects No Yes NA No No Yes No No Fracture No No No Yes Yes Yes Yes Yes Dentition abnormal Yes Yes NA Yes No Yes Yes Yes Dental caries No No No Yes Yes Yes Yes NA Bone pain NA NA NA Yes Yes Yes No Yes Growth retardation Yes Yes Yes Yes No NA NA NA Recurrent infection Yes Yes Yes No No Yes No Yes

SD, Splicing Defect; Het, heterozygous; Homo, Homozygous; ComHet, Compound Heterozygous; NA, Not Available; ms, months.

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